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Introduction Thrombin stimulation enhances plasminogen binding to platelets and promotes platelet-dependent plasmin generation. The objective of this study was to determine whether carboxyterminal lysines (C-lysines) are important for these processes, as they are in other cell types. Materials and methods 125I-plasminogen and varying concentrations of unlabeled plasminogen were added to washed platelets that were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. In some experiments the platelets were digested with carboxypeptidase B to remove C-lysines. Platelet-dependent plasmin generation was also studied by adding plasminogen and tissue plasminogen activator to platelet suspensions and monitoring the conversion of a plasmin specific chromogenic substrate. The cells were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. The effect of the thrombin inhibitor lepirudin and the plasmin inhibitor aprotinin on plasminogen binding and the appearance of C-lysines was also investigated. Results Thrombin, but not thrombin receptor activating peptide or ADP, stimulated high-affinity binding of plasminogen and greatly promoted platelet-dependent plasmin generation. Digestion with carboxypeptidase B eliminated thrombin-induced high-affinity binding and reduced thrombin-induced plasmin generation by increasing the Michaelis constant. Lepirudin, but not aprotinin, inhibited thrombin-stimulated plasminogen binding to platelets. Conclusion C-terminal lysines are necessary for high-affinity binding of plasminogen to platelets and for platelet-supported plasmin generation. The origin of the C-lysines is not clear, but they may result from a direct effect of thrombin, rather than an intermediate enzyme such as plasmin.

Long-term intravenous catheters, or venous access deviallows them to adsorb to both polar and hydrophobic ces (VADs), are widely used in the care of chronically ill patients to administer medications and blood products and to obtain blood samples for laboratory testing [1]. Although the intravascular component of VADs is made of elastomers (e.g., silicone rubber, polyurethane) selected for their physical and chemical properties to be nonreactive with blood, a major complication of VADs continues to be thrombosis [2,3]. The pathogenesis of VAD-related thrombosis is undoubtedly multifactorial, but there is evidence that the catheter material itself is thrombogenic. Within minutes, for example, the catheters adsorb coagulation proteins from whole blood [4], and adsorption of fibrinogen leads to platelet adhesion [5]. Furthermore, VADs removed from patients are rich with thrombin, which presumably supports ongoing coagulation and platelet aggregation at the catheter surface [6]. In an attempt to reduce the thrombogenicity of VADs, catheter polymers have been chemically modified to bind heparin [7,8]. Heparin was the only practical anticoagulant available for treating catheters until the direct thrombin inhibitors, recombinant hirudins and argatroban, were introduced into clinical use [9,10]. The potential for the newer anticoagulants for promoting the thromboresistance of VADs has received limited attention [7,11]. Because the direct thrombin inhibitors are proteins or amino acid derivatives rather than glycosaminoglycans, their physical interaction with the surface of catheters is different from that of heparin. The amphipathic property of proteins in particular

We have analyzed the within-subject variability of a battery of parameters of coagulant and fibrinolytic capacity and activity in postmenopausal women. We observed large differences in within-subject variability among the tests and have demonstrated how such data can be used to estimate the number of times a parameter must be measured to produce a statistically adequate sample.

intact platelet membrane glycoprotein Ib (GPIb), of platelet concentrates over a period of 6 days and which is a critical protein for binding of von Willebrand factor (vWf). The reduction in GPIb integ- have analyzed the correlation of these parameters rity results from proteolytic cleavage of glycocalicin with the changes in GC. (GC), the carbohydrate-rich amino terminus of GPIb. GC has been shown to increase in the plasma of stored platelet concentrates (PCs) [2‐4]. 1. Materials and Methods

Hemostasis is stimulated by exposure of the blood to extravascular proteins. When a blood vessel is broken, platelets quickly adhere to subendothelial collagen and von Willebrand factor, skidding onto their target like airplanes on a runway. On landing they activate and send out signals (ADP, thromboxane A2) that recruit neighboring platelets to aggregate at the site (Fig. 1). The mass of platelets, plus local vasoconstriction, provides a beachhead for hemostasis, which is quickly reinforced by integrated enzymatic reactions that change liquid blood to solid (Fig. 2). The transformation begins with tissue factor, a cell membrane protein exposed at the wound that binds trace amounts of activated factor VII (fVIIa) in the plasma. This starter is soon turned off by btissue factor pathway inhibitor,Q but not before a propagation phase of coagulation is underway that produces increasing amounts of thrombin, the enzyme that eventually converts soluble fibrinogen into solid fibrin to form a stable bhemostatic plugQ [1]. The entire process is contained at the site of vessel injury by anticoagulant proteins (antithrombin and activated protein C) that prevent the reactions from spreading abroad [2].

Introduction Thrombotic disease continues to account for significant morbidity and mortality. Ultrasound energy has been investigated as a potential primary and adjunctive treatment for thrombotic disease. We have previously shown that pulsed-high intensity focused ultrasound (HIFU) enhances thrombolysis induced by tissue plasminogen activator (tPA) in vitro , including describing the non-destructive mechanism by which tPA availability and consequent activity are increased. In this study we aimed to determine if the same effects could be achieved in vivo . Materials and methods In this study, pulsed-HIFU exposures combined with tPA boluses were compared to treatment with tPA alone, HIFU alone and control in a novel in vivo clot model. Clots were formed in the rabbit marginal ear vein and verified using venography and infrared imaging. The efficacy of thrombolytic treatment was monitored via high resolution ultrasonography for 5 h post-treatment. The cross-sectional area of clots at 4 points along the vein was measured and normalized to the pre-treatment size. Results At 5 h the complete recanalization of clots treated with pulsed-HIFU and tPA was significantly different from the partial recanalization seen with tPA treatment alone. tPA treatment alone showed a significant decrease in clot versus control, where HIFU was not significantly different than control. Histological analysis of the vessel walls in the treated veins showed no apparent irreversible damage to endothelial cells or extravascular tissue. Conclusions This study demonstrates that tPA mediated thrombolysis can be significantly enhanced when combined with non-invasive pulsed-HIFU exposures.

A frequent complication of venous access devices (VADs) is axillary-subclavian venous thrombosis. To study this problem we have compared blood drawn through VADs with peripheral blood samples in a group of oncology patients with venographically demonstrated venous damage (N = 14) and a group with normal venograms (N = 21). The samples were assayed for a battery of proteins believed to be involved in thrombogenesis. After approximately six weeks of catheterization the venographically abnormal patients had significantly less thrombomodulin (P = 0.0055) and significantly higher PAI:tPA (P = 0.022) in catheter-drawn samples as compared with the venographically normal group. Although the data are inconclusive, it is hypothesized that these changes resulted from local endothelial injury.

The binding of porcine heparin to human platelets was studied using [3H] heparin and various concentrations of unlabeled heparin. The binding was readily reversible and included saturable (Kd approximately 1.5 mg/L, R approximately 500 mg/10(15) cells) and unsaturable components. Approximately normal binding also occurred to platelets from patients with the Bernard-Soulier syndrome and Glanzmann's thrombasthenia. Competitive inhibition studies were performed with a variety of sulfated polysaccharides. Inhibitory potency correlated with charge density but not with the fundamental sugar composition of the tested compounds.

The adsorption of 125I-thrombin to polypropylene tubes has been studied with thrombin dissolved in tris-buffered saline (TBS), TBS with 0.66% polyethylene glycol (PEG), and TBS with 0.01% or 0.20% bovine serum albumin (BSA). Adsorption stabilized over time from all three solutions, increased with increasing concentrations of unadsorbed thrombin, and was reversible by repetitive washing. At equilibrium, more thrombin adsorbed from TBS-PEG than from TBS. The difference was attributable to an increased amount of thrombin deposited by evaporation during aspiration of the polypropylene tubes. Adsorption equilibrium and capacity were otherwise relatively unaffected by the presence of PEG. However, PEG was effective in retarding thrombin adsorption by markedly reducing the adsorption rate. Reduction of adsorption in the presence of BSA, on the other hand, was explained by competitive inhibition. Pretreatment of the tubes with PEG or BSA was also shown to be effective in reducing thrombin adsorption from TBS.